Comparative analysis of salivary bacterial microbiome diversity in edentulous infants and their mothers or primary care givers using pyrosequencing

Abstract

Bacterial contribution to oral disease has been studied in young children, but there is a lack of data addressing the developmental perspective in edentulous infants. Our primary objectives were to use pyrosequencing to phylogenetically characterize the salivary bacterial microbiome of edentulous infants and to make comparisons against their mothers. Saliva samples were collected from 5 edentulous infants (mean age = 4.6±1.2 mo old) and their mothers or primary care givers (mean age = 30.8±9.5 y old). Salivary DNA was extracted, used to generate DNA amplicons of the V4-V6 hypervariable region of the bacterial 16S rDNA gene, and subjected to 454-pyrosequencing. On average, over 80,000 sequences per sample were generated. High bacterial diversity was noted in the saliva of adults [1012 operational taxonomical units (OTU) at 3% divergence] and infants (578 OTU at 3% divergence). Firmicutes, Proteobacteria, Actinobacteria, and Fusobacteria were predominant bacterial phyla present in all samples. A total of 397 bacterial genera were present in our dataset. Of the 28 genera different (P<0.05) between infants and adults, 27 had a greater prevalence in adults. The exception was Streptococcus, which was the predominant genera in infant saliva (62.2% in infants vs. 20.4% in adults; P<0.05). Veillonella, Neisseria, Rothia, Haemophilus, Gemella, Granulicatella, Leptotrichia, and Fusobacterium were also predominant genera in infant samples, while Haemophilus, Neisseria, Veillonella, Fusobacterium, Oribacterium, Rothia, Treponema, and Actinomyces were predominant in adults. Our data demonstrate that although the adult saliva bacterial microbiome had a greater OTU count than infants, a rich bacterial community exists in the infant oral cavity prior to tooth eruption. Streptococcus, Veillonella, and Neisseria are the predominant bacterial genera present in infants. Further research is required to characterize the development of oral microbiota early in life and identify environmental factors that impact colonization and oral and gastrointestinal disease risk.

Figure 1. Dual hierarchical clustering dendogram of the bacterial phyla profiles for babies and mothers.

Baby and mother number and sequence direction [forward (F); reverse(R)] are provided on the bottom x-axis. The dendogram provides the phylum designation along the right y-axis and the abundance relationship across all samples for each phylum based upon Wards clustering and Manhattan distance methods along the left y-axis. The relative distance scale for the left y-axis is provided in the lower left corner of the figure. The samples have also been clustered based upon Wards and Manhattan principles with sample designations along the bottom x-axis and the clustering described along the top x-axis in the figure. The heatmap depicts the relative percentage of each phyla for each sample. The color scale for the heatmap is shown in the upper left corner of the figure. It is obvious from the figure that the microbiome of the mothers and babies are clearly separable but forward and reverse reads cluster together.

Figure 2. Dual hierarchical clustering dendogram of the most predominant and ubiquitous 50 bacterial genera among the samples.

Baby and mother number and sequence direction [forward (F); reverse(R)] are provided on the bottom x-axis. Those genera that occurred in only 1 sample were omitted, along with those present in relative average percentages<0.09% in 3 or fewer samples. The genera that occurred in average percentages <0.01% among all samples were also excluded. It should be noted that inclusion of all genera did not change the clustering patterns (data not shown), thus this analysis accurately represents and overview and relationship among samples for the microbiome data. The dendogram provides the genus designation along the right y axis and the abundance relationship across all samples for each genus based upon Wards clustering and Manhattan distance methods along the left y-axis. The relative distance scale for the left y-axis is provided in the lower left corner of the figure. The samples have also been clustered based upon Wards and Manhattan principles with sample designations along the bottom x-axis and the clustering described along the top x-axis in the figure. The heatmap depicts the relative percentage of each genus for each sample. The color scale for the heatmap is shown in the upper left corner of the figure.

Figure 3. Principal component analysis of UNIFRAC distance metric.

This figure provides a 3 dimensional visualization of the PCA analysis using the top 3 vectors. Red balls representing the mother samples and blue balls representing the baby samples clearly show the significant clustering distance between the two groups. Eigenvalues and % variation explained by each vector are Vector 1 eigval = 0.211 (41.5%), Vector 2 = 0.137 (26.8%), and Vector 3 0.061 (12%).